Biomarkers of Embryo Quality
Study Question Could cell-free DNA (cfDNA) quantification in individual human follicular fluid (FF) samples become a new non-invasive predictive biomarker for in vitro fertilization (IVF) outcomes?
Summary Answer CfDNA level in human follicular fluid samples was significantly correlated with embryo quality and could be used as an innovative non-invasive biomarker to improve IVF outcomes.
What is Known Already CfDNA fragments, resulting from apoptotic or necrotic events, are present in the bloodstream and their quantification is already used as a biomarker for gynaecological and pregnancy disorders. Follicular fluid is important for oocyte development and contains plasma components and factors secreted by granulosa cells during folliculogenesis. CfDNA presence in follicular fluid and its potential use as an IVF outcome biomarker have never been investigated.
Study Design, Size, Duration One hundred individual follicular fluid samples were collected from 43 female patients undergoing conventional IVF (n = 26) or ICSI (n = 17). CfDNA level was quantified in each individual follicular fluid sample.
Participants/Materials, Setting, Methods At oocyte collection day, follicles were aspirated individually. Only blood-free follicular fluid samples were included in the study. Follicle size was calculated based on the follicular fluid volume. Each corresponding cumulus-oocyte complex was isolated for IVF or ICSI procedures. Follicular fluid cfDNA was measured by quantitative PCR with ALU-specific primers.
Main Results and the Role of Chance Human follicular fluid samples from individual follicles contain measurable amounts of cfDNA (mean ± SD, 1.62 ± 2.08 ng/µl). CfDNA level was significantly higher in small follicles (8–12 mm in diameter) than in large ones (>18 mm) (mean ± SD, 2.54 ± 0.78 ng/µl versus 0.71 ± 0.44 ng/µl, respectively, P = 0.007). Moreover, cfDNA concentration was significantly and negatively correlated with follicle size (r = −0.34; P = 0.003). A weak significant negative correlation between DNA integrity and 17β-estradiol level in follicular fluid samples at oocyte collection day was observed (r = −0.26; P = 0.008). CfDNA level in follicular fluid samples corresponding to top quality embryos was significantly lower than in follicular fluid samples related to poor quality embryos (P = 0.022). Similarly, cfDNA level was also significantly lower in follicular fluid samples related to embryos with low fragmentation rate (≤25%) than with high fragmentation rate (>25%) (P = 0.02).
Limitations, Reasons for Caution A larger study should be conducted in order to establish the predictive value of cfDNA level for embryo quality and to investigate whether follicular fluid cfDNA levels are correlated with embryo implantation rates and pregnancy outcomes. Moreover, the role of follicular fluid cfDNA on embryo quality should be studied to determine whether high cfDNA concentration in follicular fluid is only a consequence or also a cause of follicular dysfunction.
Wider Implications of the Findings CfDNA evaluation in individual follicular fluid samples might represent an innovative biomarker of embryo quality to use as a supplemental tool to predict embryo quality during IVF.
Study Funding/Competing Interest(S) This study was partially supported by the University Hospital of Montpellier and Ferring Pharmaceuticals. The authors of the study have no competing interests to report.
Currently, selection of the embryos with the highest implantation potential during assisted reproductive technology (ART) procedures relies only on morphological criteria. A new method based on time-lapse imaging has been recently described for the acquisition of embryo morphokinetic data to help such selection (Meseguer et al., 2011; Herrero and Meseguer, 2013). Nevertheless, the subjective observation of embryo morphology to predict a successful pregnancy shows limitations (Guerif et al., 2007; Assou et al., 2008, 2010; Aydiner et al., 2010). Therefore, many recent works have focused on the identification of new non-invasive biomarkers based on the analysis of the oocyte microenvironment to improve the accuracy of embryo selection (Pearson, 2006; Assou et al., 2008, 2010; van Montfoort et al., 2008; Aydiner et al., 2010; Uyar et al., 2013). In some studies, follicular fluid (FF) components, which are derived from plasma or secreted from granulosa cells, were investigated as potential biomarkers (Baka and Malamitsi-Puchner, 2006; De Placido et al., 2006; Yanaihara et al., 2007; Estes et al., 2009; Revelli et al., 2009; Borowiecka et al., 2012; Lédée et al., 2013). Indeed, follicular fluid, which surrounds the oocyte, is involved in follicular maturation, oocyte growth and the gradual acquisition of developmental competence. Consequently, follicular fluid might represent a reliable source of oocyte and embryo outcome biomarkers that could be used as supplemental prognostic/diagnostic tools in ART (Mermillod et al., 1999; Mendoza et al., 2002; Sutton et al., 2003; Krisher, 2004; Angelucci et al., 2006; Baka and Malamitsi-Puchner, 2006).
Cell-free DNA (cfDNA) fragments can be detected in the bloodstream (Mandel and Métais, 1948; Swarup and Rajeswari, 2007) and result from apoptotic or necrotic processes. They are released via a passive or an active mechanism (Jahr et al., 2001; Stroun et al., 2001). Nuclear and mitochondrial DNA can be released passively in the blood from apoptotic or necrotic cells (Schwarzenbach et al., 2011a,b) and are then phagocytized by macrophages in healthy individuals, in whom the basal cfDNA level remains low (Jiang et al., 2003; Pisetsky and Fairhurst, 2007). CfDNA can also be actively secreted by cells (Gahan et al., 2008), leading to an increase of cfDNA circulating level in some cancers and other serious disorders. For that reason, cfDNA is used as non-invasive diagnostic and/or prognostic biomarker for some cancers and other severe pathologies (Paci et al., 2009; Gao et al., 2010; Kamat et al., 2010; Vlassov et al., 2010; Schwarzenbach et al., 2011a,b; Gahan, 2012; Chen et al., 2013; da Silva Filho et al., 2013). Similarly, the emergence of non-invasive prenatal testing, based on fetal cfDNA detection in the maternal blood, constitutes a promising approach in obstetrics and gynaecology (Wright and Burton, 2009; Liao et al., 2014). So far, no study has evaluated cfDNA content in ovarian follicles, although follicular atresia is the result of many apoptotic events in granulosa cells.
In this study, we asked whether cfDNA could be detected in follicular fluid and whether its quantification could be used to develop an innovative prognostic test for embryo quality. To this aim, we quantified cfDNA in the follicular fluid of individual follicles from patients undergoing conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). We then explored whether cfDNA level or integrity was related to follicle size or hormonal content in follicular fluid. Moreover, we investigated the relationship between cfDNA levels and patients' clinical characteristics and IVF outcomes, respectively. Our findings suggest that cfDNA quantification in follicular fluid could represent an innovative and non-invasive biomarker to improve embryo selection in IVF procedures.
Abstract and Introduction
Abstract
Study Question Could cell-free DNA (cfDNA) quantification in individual human follicular fluid (FF) samples become a new non-invasive predictive biomarker for in vitro fertilization (IVF) outcomes?
Summary Answer CfDNA level in human follicular fluid samples was significantly correlated with embryo quality and could be used as an innovative non-invasive biomarker to improve IVF outcomes.
What is Known Already CfDNA fragments, resulting from apoptotic or necrotic events, are present in the bloodstream and their quantification is already used as a biomarker for gynaecological and pregnancy disorders. Follicular fluid is important for oocyte development and contains plasma components and factors secreted by granulosa cells during folliculogenesis. CfDNA presence in follicular fluid and its potential use as an IVF outcome biomarker have never been investigated.
Study Design, Size, Duration One hundred individual follicular fluid samples were collected from 43 female patients undergoing conventional IVF (n = 26) or ICSI (n = 17). CfDNA level was quantified in each individual follicular fluid sample.
Participants/Materials, Setting, Methods At oocyte collection day, follicles were aspirated individually. Only blood-free follicular fluid samples were included in the study. Follicle size was calculated based on the follicular fluid volume. Each corresponding cumulus-oocyte complex was isolated for IVF or ICSI procedures. Follicular fluid cfDNA was measured by quantitative PCR with ALU-specific primers.
Main Results and the Role of Chance Human follicular fluid samples from individual follicles contain measurable amounts of cfDNA (mean ± SD, 1.62 ± 2.08 ng/µl). CfDNA level was significantly higher in small follicles (8–12 mm in diameter) than in large ones (>18 mm) (mean ± SD, 2.54 ± 0.78 ng/µl versus 0.71 ± 0.44 ng/µl, respectively, P = 0.007). Moreover, cfDNA concentration was significantly and negatively correlated with follicle size (r = −0.34; P = 0.003). A weak significant negative correlation between DNA integrity and 17β-estradiol level in follicular fluid samples at oocyte collection day was observed (r = −0.26; P = 0.008). CfDNA level in follicular fluid samples corresponding to top quality embryos was significantly lower than in follicular fluid samples related to poor quality embryos (P = 0.022). Similarly, cfDNA level was also significantly lower in follicular fluid samples related to embryos with low fragmentation rate (≤25%) than with high fragmentation rate (>25%) (P = 0.02).
Limitations, Reasons for Caution A larger study should be conducted in order to establish the predictive value of cfDNA level for embryo quality and to investigate whether follicular fluid cfDNA levels are correlated with embryo implantation rates and pregnancy outcomes. Moreover, the role of follicular fluid cfDNA on embryo quality should be studied to determine whether high cfDNA concentration in follicular fluid is only a consequence or also a cause of follicular dysfunction.
Wider Implications of the Findings CfDNA evaluation in individual follicular fluid samples might represent an innovative biomarker of embryo quality to use as a supplemental tool to predict embryo quality during IVF.
Study Funding/Competing Interest(S) This study was partially supported by the University Hospital of Montpellier and Ferring Pharmaceuticals. The authors of the study have no competing interests to report.
Introduction
Currently, selection of the embryos with the highest implantation potential during assisted reproductive technology (ART) procedures relies only on morphological criteria. A new method based on time-lapse imaging has been recently described for the acquisition of embryo morphokinetic data to help such selection (Meseguer et al., 2011; Herrero and Meseguer, 2013). Nevertheless, the subjective observation of embryo morphology to predict a successful pregnancy shows limitations (Guerif et al., 2007; Assou et al., 2008, 2010; Aydiner et al., 2010). Therefore, many recent works have focused on the identification of new non-invasive biomarkers based on the analysis of the oocyte microenvironment to improve the accuracy of embryo selection (Pearson, 2006; Assou et al., 2008, 2010; van Montfoort et al., 2008; Aydiner et al., 2010; Uyar et al., 2013). In some studies, follicular fluid (FF) components, which are derived from plasma or secreted from granulosa cells, were investigated as potential biomarkers (Baka and Malamitsi-Puchner, 2006; De Placido et al., 2006; Yanaihara et al., 2007; Estes et al., 2009; Revelli et al., 2009; Borowiecka et al., 2012; Lédée et al., 2013). Indeed, follicular fluid, which surrounds the oocyte, is involved in follicular maturation, oocyte growth and the gradual acquisition of developmental competence. Consequently, follicular fluid might represent a reliable source of oocyte and embryo outcome biomarkers that could be used as supplemental prognostic/diagnostic tools in ART (Mermillod et al., 1999; Mendoza et al., 2002; Sutton et al., 2003; Krisher, 2004; Angelucci et al., 2006; Baka and Malamitsi-Puchner, 2006).
Cell-free DNA (cfDNA) fragments can be detected in the bloodstream (Mandel and Métais, 1948; Swarup and Rajeswari, 2007) and result from apoptotic or necrotic processes. They are released via a passive or an active mechanism (Jahr et al., 2001; Stroun et al., 2001). Nuclear and mitochondrial DNA can be released passively in the blood from apoptotic or necrotic cells (Schwarzenbach et al., 2011a,b) and are then phagocytized by macrophages in healthy individuals, in whom the basal cfDNA level remains low (Jiang et al., 2003; Pisetsky and Fairhurst, 2007). CfDNA can also be actively secreted by cells (Gahan et al., 2008), leading to an increase of cfDNA circulating level in some cancers and other serious disorders. For that reason, cfDNA is used as non-invasive diagnostic and/or prognostic biomarker for some cancers and other severe pathologies (Paci et al., 2009; Gao et al., 2010; Kamat et al., 2010; Vlassov et al., 2010; Schwarzenbach et al., 2011a,b; Gahan, 2012; Chen et al., 2013; da Silva Filho et al., 2013). Similarly, the emergence of non-invasive prenatal testing, based on fetal cfDNA detection in the maternal blood, constitutes a promising approach in obstetrics and gynaecology (Wright and Burton, 2009; Liao et al., 2014). So far, no study has evaluated cfDNA content in ovarian follicles, although follicular atresia is the result of many apoptotic events in granulosa cells.
In this study, we asked whether cfDNA could be detected in follicular fluid and whether its quantification could be used to develop an innovative prognostic test for embryo quality. To this aim, we quantified cfDNA in the follicular fluid of individual follicles from patients undergoing conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). We then explored whether cfDNA level or integrity was related to follicle size or hormonal content in follicular fluid. Moreover, we investigated the relationship between cfDNA levels and patients' clinical characteristics and IVF outcomes, respectively. Our findings suggest that cfDNA quantification in follicular fluid could represent an innovative and non-invasive biomarker to improve embryo selection in IVF procedures.